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Secreted factors from cultured dental pulp stem cells promoted neurite outgrowth of dorsal root ganglion neurons and ameliorated neural functions in streptozotocin-induced diabetic mice.

ABSTRACT: AIMS/INTRODUCTION:Transplantation of stem cells promotes axonal regeneration and angiogenesis in a paracrine manner. In the present study, we examined whether the secreted factors in conditioned medium of stem cells from human exfoliated deciduous teeth (SHED-CM) had beneficial effects on diabetic polyneuropathy in mice. MATERIALS AND METHODS:Conditioned medium of stem cells from human exfoliated deciduous teeth was collected 48 h after culturing in serum-free Dulbecco's modified Eagle's medium (DMEM), and separated into four fractions according to molecular weight. Dorsal root ganglion neurons from C57BL/6J mice were cultured with SHED-CM or DMEM to evaluate the effect on neurite outgrowth. Streptozotocin-induced diabetic mice were injected with 100 ?L of SHED-CM or DMEM into the unilateral hindlimb muscles twice a week over a period of 4 weeks. Peripheral nerve functions were evaluated by the plantar test, and motor and sensory nerve conduction velocities. Intraepidermal nerve fiber densities, capillary number-to-muscle fiber ratio, capillary blood flow and morphometry of sural nerves were also evaluated. RESULTS:Conditioned medium of stem cells from human exfoliated deciduous teeth significantly promoted neurite outgrowth of dorsal root ganglion neurons compared with DMEM. Among four fractions of SHED-CM, the only fraction of <6 kDa promoted the neurite outgrowth of dorsal root ganglion neurons. In addition, SHED-CM significantly prevented decline in sensory nerve conduction velocities compared with DMEM in diabetic mice. Although SHED-CM did not improve intraepidermal nerve fiber densities or morphometry of sural nerves, SHED-CM ameliorated the capillary number-to-muscle fiber ratio and capillary blood flow. CONCLUSIONS:These results suggested that SHED-CM might have a therapeutic effect on diabetic polyneuropathy through promoting neurite outgrowth, and the increase in capillaries might contribute to the improvement of neural function.

SUBMITTER: Miura-Yura E

PROVIDER: S-EPMC6944849 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Secreted factors from cultured dental pulp stem cells promoted neurite outgrowth of dorsal root ganglion neurons and ameliorated neural functions in streptozotocin-induced diabetic mice.

Miura-Yura Emiri E Tsunekawa Shin S Naruse Keiko K Nakamura Nobuhisa N Motegi Mikio M Nakai-Shimoda Hiromi H Asano Saeko S Kato Makoto M Yamada Yuichiro Y Izumoto-Akita Takako T Yamamoto Akihito A Himeno Tatsuhito T Kondo Masaki M Kato Yoshiro Y Nakamura Jiro J Kamiya Hideki H

Journal of diabetes investigation 20190621 1

<h4>Aims/introduction</h4>Transplantation of stem cells promotes axonal regeneration and angiogenesis in a paracrine manner. In the present study, we examined whether the secreted factors in conditioned medium of stem cells from human exfoliated deciduous teeth (SHED-CM) had beneficial effects on diabetic polyneuropathy in mice.<h4>Materials and methods</h4>Conditioned medium of stem cells from human exfoliated deciduous teeth was collected 48 h after culturing in serum-free Dulbecco's modified ...[more]

PMID: 31144464

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Project description:Transforming growth factor (TGF) plays an important role in tooth morphogenesis and mineralization. During postnatal development, the dental pulp (DP) mesenchyme secretes neurotrophic factors that guide trigeminal nerve fibers into and throughout the DP. This process is tightly linked with dentin formation and mineralization. Our laboratory established a mouse model in which Tgfbr2 was conditionally deleted in DP mesenchyme using an Osterix promoter-driven Cre recombinase (Tgfbr2cko). These mice survived postnatally with significant defects in bones and teeth, including reduced mineralization and short roots. Hematoxylin and eosin staining revealed reduced axon-like structures in the mutant mice. Reporter imaging demonstrated that Osterix-Cre activity within the tooth was active in the DP and derivatives, but not in neurons. Immunofluorescence staining for 3 tubulin (neuronal marker) was performed on serial cryosections from control and mutant molars on postnatal days 7 and 24 (P7, P24). Confocal imaging and pixel quantification demonstrated reduced innervation in Tgfbr2cko first molars at both stages compared to controls, indicating that signals necessary to promote neurite outgrowth were disrupted by Tgfbr2 deletion. We performed mRNA-Sequence (RNA-Seq) and gene onotology analyses using RNA from the DP of P7 control and mutant mice to investigate the pathways involved in Tgfbr2-mediated tooth development. These analyses identified downregulation of several mineralization-related and neuronal genes in the Tgfbr2cko DP compared to controls. Select gene expression patterns were confirmed by quantitative real-time PCR and immunofluorescence imaging. Lastly, trigeminal neurons were co-cultured atop Transwell filters overlying primary Tgfbr2f/f DP cells. Tgfbr2 in the DP was deleted via adenovirus-expressed Cre recombinase. Confocal imaging of axons through the filter pores showed increased axonal sprouting from neurons cultured with Tgfbr2-positive DP cells compared to neurons cultured alone. Axon sprouting was reduced when Tgfbr2 was knocked down in the DP cells. Immunofluorescence of dentin sialophosphoprotein in co-cultured DP cells confirmed reduced mineralization potential in cells with Tgfbr2 deletion. Both our proteomics and RNA-Seq analyses indicate that axonal guidance cues, particularly semaphorin signaling, were disrupted by Tgfbr2 deletion. Thus, Tgfbr2 in the DP mesenchyme appears to regulate differentiation and the cells’ ability to guide neurite outgrowth during tooth mineralization and innervation.

Dataset Information

Secreted factors from cultured dental pulp stem cells promoted neurite outgrowth of dorsal root ganglion neurons and ameliorated neural functions in streptozotocin-induced diabetic mice.

Publications

Secreted factors from cultured dental pulp stem cells promoted neurite outgrowth of dorsal root ganglion neurons and ameliorated neural functions in streptozotocin-induced diabetic mice.

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